The flaviviruses are mosquito-borne viruses that are associated with significant worldwide morbidity and mortality. The dengue viruses (DENV) are members of this group associated with dengue fever, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). These viruses are endemic in most of the tropical and sub-tropical world, and roughly one-third of the Earth's populace lives in areas at risk for dengue transmission. A second member of the flaviviruses. West Nile virus (WNV), is a neurotropic virus; infection can result in encephalitis/meningitis and subsequent long-term neurologic complications or death. Despite this threat, no specific therapy exists for either virus. Treatment is largely supportive, and in the case of DHF and DSS or neuroinvasive WNV, generally requires hospitalization. In this application, we propose to identify and develop small molecule inhibitors of these viruses. We will emphasize the development of molecules that show broad-spectrum activity against multiple flaviviruses, and potentially members of other virus families as well. Initial screening of multiple compound libraries comprising >100,000 compounds will be carried out in collaboration with the Southern Research Institute (SRI). In additional arms of this proposal, the same libraries will be screened for activity against other virus families, including influenza, alphaviruses, and coronaviruses. Therefore, the overall proposal has the potential to identify compounds that are not only active against flaviviruses, but other medically important viruses as well. Multiple techniques will be used to characterize mechanisms of action and targets of antiviral compounds. We will focus on compounds that target one of the following important enzymatic activities of the flavivirus NS5 protein: the RNA-dependent RNA polymerase, which is essential for replication of the viral RNA genome, and the 2'-0-methyltransferase, which is required for the virus to avoid the host innate immune response. Compounds with activities targeting these enzymes will be further developed through the synthesis of structural analogs, analysis of structure activity relationship (SAR), and testing in in vivo (mouse) models of virus infection.